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Gomez-Skarmeta, J. L.

Publications and source records attributed to Gomez-Skarmeta, J. L..

At least 19 recordsLinked to original sources

Deep conservation of cis-regulatory elements and chromatin organization in echinoderms uncover ancestral regulatory features of animal genomes

Despite the growing abundance of sequenced animal genomes, we only have detailed knowledge of regulatory organization for a handful of lineages, particularly flies and vertebrates. These two groups of taxa show contrasting trends in the molecular mechanisms of 3D chromatin organization and long-term evolutionary dynamics of cis-regulatory element (CREs) conservation. To help us identify shared versus derived features that could be responsible for the evolution of these different regulatory architectures in animals, we studied the evolution and organization of the regulatory genome of echinoderms, a lineage whose phylogenetic position and relatively slow molecular evolution has proven particularly useful for evolutionary studies. First, using PacBio and HiC data, we generated new reference genome assemblies for two species belonging to two different echinoderm classes: the purple sea urchin Strongylocentrotus purpuratus and the bat sea star Patiria miniata. Second, we characterized their 3D chromatin architecture, identifying TAD-like domains in echinoderms that, like in flies, do not seem to be associated with CTCF motif orientation. Third, we systematically profiled CREs during sea star and sea urchin development using ATAC-seq, comparing their regulatory logic and dynamics over multiple developmental stages. Finally, we investigated sea urchin and sea star CRE evolution across multiple evolutionary distances and timescales, from closely related species to other echinoderm classes and deuterostome lineages. This showed the presence of several thousand elements conserved for hundreds of millions of years, revealing a vertebrate-like pattern of CRE evolution that probably constitutes an ancestral property of the regulatory evolution of animals.

evolutionary biology↗

An amphioxus neurula stage cell atlas supports a complex scenario for the emergence of vertebrate head mesoderm

The emergence of new structures can often be linked to the evolution of novel cell types that follows the rewiring of developmental gene regulatory subnetworks. Vertebrates are characterized by a complex body plan compared to the other chordate clades and the question remains of whether and how the emergence of vertebrate morphological innovations can be related to the appearance of new embryonic cell populations. We already proposed, by studying mesoderm development in the cephalochordate amphioxus, a scenario for the evolution of the vertebrate head mesoderm. To further test this scenario at the cell population level, we used scRNA-seq to construct a cell atlas of the amphioxus neurula, stage at which the main mesodermal compartments are specified. Our data allowed us to confirm the presence of a prechordal-plate like territory in amphioxus, and shows that cell populations of the anteriormost somites and of the ventral part of the somites present a transcriptomic profile supporting the homology with vertebrate cranial/pharyngeal and lateral plate mesoderm. Finally, our work provides evidence that the appearance of the specific mesodermal structures of the vertebrate head was associated to both segregation of pre-existing cell populations, and co-option of new genes for the control of myogenesis.

evolutionary biology↗

Comparative 3D genome analysis between neural retina and RPE reveals differential cis-regulatory interactions at retinal disease loci

Vision depends on the functional interplay between the photoreceptor cells of the neural retina and the supporting cells of the underlying retinal pigment epithelium (RPE). Most genes involved in inherited retinal diseases (IRD) display highly specific spatiotemporal expression within these interconnected retinal components through the local recruitment of cis-regulatory elements (CREs) in 3D nuclear space. To understand the role of differential chromatin architecture in establishing tissue-specific expression patterns at IRD loci in the human neural retina and the RPE, we mapped genome-wide chromatin interactions by applying in situ Hi-C and H3K4me3 HiChIP to human adult post-mortem donor retinas. A comparative 3D genome analysis between neural retina and RPE/choroid revealed that almost 60% of 290 known IRD genes were marked by differential 3D genome structure and/or cis-regulatory interactions. One of these genes was ABCA4, which is implicated in the most common autosomal recessive IRD. We zoomed in on tissue-specific chromatin interactions at the ABCA4 locus using high-resolution UMI-4C assays. Upon integration with bulk and single-cell epigenomic datasets and in vivo enhancer assays in zebrafish, we revealed tissue-specific CREs interacting with ABCA4. In summary, through extensive comparative 3D genome mapping, based on genome-wide (Hi-C), promoter-centric (HiChIP) and locus-specific (UMI-4C) assays of human neural retina and RPE, we have shown that gene regulation at key IRD loci is likely mediated by tissue-specific chromatin interactions. These findings do not only provide insight into tissue-specific regulatory landscapes of IRD genes, but also delineate the search space for non-coding genomic variation underlying unsolved IRD. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/543842v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@fd0d7dorg.highwire.dtl.DTLVardef@180a5bforg.highwire.dtl.DTLVardef@101aec1org.highwire.dtl.DTLVardef@1781d1d_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

Rewiring of the epigenome and chromatin architecture by retinoic acid signaling during zebrafish embryonic development

BackgroundRetinoic acid (RA) functions as a ligand for the nuclear RA receptors (RARs), which regulate the expression of target genes by binding to RA response elements. RA signaling is required for multiple processes during chordate embryonic development, such as body axis extension, hindbrain antero-posterior patterning and forelimb bud initiation. Although some RA target genes have been identified, little is known about the genome-wide effects of RA signaling during in vivo embryonic development. ResultsHere we stimulate the RA pathway during development by treating zebrafish embryos with all-trans-RA (atRA), the most abundant form of RA, and use a combination of RNA-seq, ATAC-seq, ChIP-seq and HiChIP to gain insight into the molecular mechanisms by which RA signaling control target gene expression. We find that RA signaling is involved in anterior/posterior patterning and development of the central nervous system, participating in the transition from pluripotency to differentiation. atRA treatment also induces alterations in chromatin accessibility during early development and promotes chromatin binding of RARa and the RA targets Hoxb1b, Meis2b and Sox3, which cooperate in central nervous system development. Finally, we show that RA induces a rewiring of chromatin architecture, with alterations in chromatin 3D interactions that are consistent with target gene expression. This is illustrated by the specific induction of anterior HoxB genes by RARs, among other examples. ConclusionsAltogether, our findings identify genome-wide targets of RA signaling during embryonic development and provide a molecular mechanism by which developmental signaling pathways regulate the expression of target genes by altering chromatin topology.

developmental biology↗

An integrative multi-omics approach to draft gene regulatory networks at a cell type resolution

Drafting gene regulatory networks (GRNs) requires embryological knowledge pertaining to the cell type families, information on the regulatory genes, causal data from gene knockdown experiments and validations of the identified interactions by cis-regulatory analysis. We use multi-omics involving next-generation sequencing (-seq) to obtain the necessary information drafting the sea urchin posterior gut GRN. Here we present an update to the GRN using i) a single cell RNA-seq derived cell atlas highlighting the 2 day post fertilization (dpf) sea urchin gastrula cell type families, as well as the genes expressed at single cell level, ii) a set of putative cis-regulatory modules and transcription factor (TF) binding sites obtained from chromatin accessibility ATAC-seq data, and iii) interactions directionality obtained from differential bulk RNA-seq following knockdown of the TF Sp-Pdx1, a key regulator of gut patterning in sea urchins. Combining these datasets, we draft the GRN for the hindgut Sp-Pdx1 positive cells in the 2 dpf gastrula embryo. Overall, our data resolves the complex connectivity of the posterior gut GRN and increases the resolution of gene regulatory cascades operating within it.

developmental biology↗

Insights into deuterostome evolution from the biphasic transcriptional programmes of hemichordates

Evolutionary history of deuterostomes remains unsolved and is intimately related to the origin of chordates. Among deuterostomes, hemichordates and echinoderms (collectively called Ambulacraria) are sister groups of chordates. Comparative studies involving these three groups provide valuable insights into deuterostome evolution. Indirect developing hemichordates produce planktonic larvae that bear resemblance to echinoderm larvae before undergoing metamorphosis into an adult body plan with anteroposterior polarity homologous to that of chordates. Therefore, understanding the developmental processes of indirect-developing hemichordates can help understand the evolution of deuterostomes and the origins of chordates. In this study, we analysed the transcriptomes and chromatin accessibility of multiple developmental stages of the indirect-developing hemichordate Ptychodera flava and discovered that it exhibits a biphasic developmental program controlled by distinct sets of transcription factors and their corresponding regulatory elements. Comparative analyses of transcriptomes and network analyses revealed that the gastrula transcriptome is relatively ancient, and the TFs orchestrating its gene expression are highly interconnected in networks of cis-regulatory interactions. Comparing the developmental transcriptomes of hemichordates, echinoderms, and amphioxus, revealed high conservation of gene expression during gastrulation that extends to the neurula stages of amphioxus, along with remarkable similarity in larval transcriptomes across the three species. Additionally, we show that P. flava possesses conserved interactions of transcription factors necessary for the development of echinoderm endomesoderm and chordate axial mesoderm, including conserved cis-regulatory elements of the FoxA transcription factor that is central to the two networks. These findings suggest the existence of a deuterostome phylotypic stage during gastrulation governed by gene regulatory networks with conserved cis-regulatory interactions. Conversely, integration of gene expression data with synteny data revealed that gene expression recapitulates the independent evolutionary history of the Ancestral Linkage Groups that underwent rearrangements in each deuterostome lineage, suggesting a potential role of genome rearrangement during the evolution of larval strategies in hemichordates and deuterostome body plans.

evolutionary biology↗

The little skate genome and the evolutionary emergence of wing-like fin appendages

Skates are cartilaginous fish whose novel body plan features remarkably enlarged wing-like pectoral fins that allow them to thrive in benthic environments. The molecular underpinnings of this unique trait, however, remain elusive. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins - gene expression, chromatin occupancy and three-dimensional (3D) conformation - we find skate-specific genomic rearrangements that alter the 3D regulatory landscape of genes involved in the planar cell polarity (PCP) pathway. Functional inhibition of PCP signaling resulted in marked reduction of anterior fin size, confirming this pathway as a major contributor of batoid fin morphology. We also identified a fin-specific enhancer that interacts with 3 HOX genes, consistent with the redeployment of Hox gene expression in anterior pectoral fins, and confirmed the potential of this element to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganizations and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.

evolutionary biology↗

Multi-omics profiling, in vitro and in vivo enhancer assays dissect the cis -regulatory mechanisms underlying North Carolina macular dystrophy, a retinal enhanceropathy

North Carolina macular dystrophy (NCMD) is a rare autosomal dominant disease affecting macular development. The disease is caused by non-coding single nucleotide variants (SNVs) in two hotspot regions near PRDM13 and by duplications in two distinct chromosomal loci, overlapping DNase I hypersensitive sites near either PRDM13 or IRX1. To unravel the mechanisms by which these variants cause disease, we first established a genome-wide multi-omics retinal database, RegRet. Integration of UMI-4C profiles we generated on adult human retina then allowed fine-mapping of the interactions of the PRDM13 and IRX1 gene promoters, and the identification of eighteen candidate cis-regulatory elements (cCREs), the activity of which was investigated by luciferase and Xenopus enhancer assays. Next, luciferase assays showed that the non-coding SNVs located in the two hotspot regions of PRDM13 affect cCRE activity, including two novel NCMD-associated non-coding SNVs that we identified. Interestingly, the cCRE containing one of these SNVs was shown to interact with the PRDM13 promoter, demonstrated in vivo activity in Xenopus, and is active at the developmental stage when progenitor cells of the central retina exit mitosis, putting forward this region as a PRDM13 enhancer. Finally, mining of single-cell transcriptional data of embryonic and adult retina revealed the highest expression of PRDM13 and IRX1 when amacrine cells start to synapse with retinal ganglion cells, supporting the hypothesis that altered PRDM13 or IRX1 expression impairs interactions between these cells during retinogenesis. Overall, this study gained insight into the cis-regulatory mechanisms of NCMD and supports that this condition is a retinal enhanceropathy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/481329v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@4a85e3org.highwire.dtl.DTLVardef@9bfe55org.highwire.dtl.DTLVardef@156a9d8org.highwire.dtl.DTLVardef@a8cb92_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

Parallel evolution of amphioxus and vertebrate small-scale gene duplications

BackgroundAmphioxus are non-vertebrate chordates characterized by a slow morphological and molecular evolution. They share the basic chordate body-plan and genome organization with vertebrates but lack their 2R whole-genome duplications and their developmental complexity. For these reasons, amphioxus are frequently used as an outgroup to study vertebrate genome evolution and Evo-Devo. Aside from whole-genome duplications, genes continuously duplicate on a smaller scale. Small-scale duplicated genes can be found in both amphioxus and vertebrate genomes, while only the vertebrate genomes have duplicated genes product of their 2R whole-genome duplications. Here, we explore the history of small-scale gene duplications in the amphioxus lineage and compare it to small- and large-scale gene duplication history in vertebrates. ResultsWe present a study of the European amphioxus (Branchiostoma lanceolatum) gene duplications thanks to a new, high-quality genome reference. We find that, despite its overall slow molecular evolution, the amphioxus lineage has had a history of small-scale duplications similar to the one observed in vertebrates. We find parallel gene duplication profiles between amphioxus and vertebrates, and conserved functional constraints in gene duplication. Moreover, amphioxus gene duplicates show levels of expression and patterns of functional specialization similar to the ones observed in vertebrate duplicated genes. We also find strong conservation of gene synteny between two distant amphioxus species, B. lanceolatum and B. floridae, with two major chromosomal rearrangements. ConclusionsIn contrast to their slower molecular and morphological evolution, amphioxus small-scale gene duplication history resembles that of the vertebrate lineage both in quantitative and in functional terms.

evolutionary biology↗

Active DNA demethylation of developmental cis-regulatory regions predates vertebrate origins

DNA methylation (5-methylcytosine; 5mC) is a repressive gene-regulatory mark required for vertebrate embryogenesis. Genomic 5mC is tightly regulated through the coordinated action of DNA methyltransferases, which deposit 5mC, and TET enzymes, which participate in its active removal through the formation of 5-hydroxymethylcytosine (5hmC). TET enzymes are essential for mammalian gastrulation and activation of vertebrate developmental enhancers, however, to date, a clear picture of 5hmC function, abundance, and genomic distribution in non-vertebrate lineages is lacking. By employing base-resolution 5mC and 5hmC quantification during sea urchin and lancelet embryogenesis, we shed light on the roles of non-vertebrate 5hmC and TET enzymes. We find that these invertebrate deuterostomes employ TET enzymes for targeted demethylation of regulatory regions associated with developmental genes and show that the complement of identified 5hmC-regulated genes is conserved to vertebrates. This work thus demonstrates that active 5mC removal from regulatory regions is a common feature of deuterostome embryogenesis suggestive of unexpected deep conservation of a major gene-regulatory module.

genomics↗

Genomic analyses reveal FoxG as an upstream regulator of wnt1 required for posterior identity specification in planarians

Embryonic specification of the first body axis requires the formation of an Organizer, a group of cells with the ability to instruct fates in the surrounding tissue. The existence of organizing regions in adults, i.e. during regeneration, which also requires patterning of new tissues, remains unstudied. To that aim, we study regeneration in planarians, flatworms that can regenerate any missing structure, even the head, in a few days. In planarians, as described in embryonic models, the cWNT pathway specifies the anterior-posterior axis. During the first 12-24h after amputation both wnt1 and notum (a Wnt inhibitor) are expressed in any wound, but 48 hours later they become restricted to posterior or anterior facing wounds, forming the anterior and the posterior organizers, respectively. In this study we undertook a genomic approach to further understand the mechanism that triggers the early expression of wnt1 and the specification of the posterior identity. Through ATAC-sequencing and CHIPmentation techniques we uncovered Cis-Regulatory Elements of Schmidtea mediterranea genome and analyzed them in notum and wnt1 (RNAi) animals. The result shows that already at 12 hours after amputation the chromatin structure of the wounds has changed its conformation according to the polarity of the pre-existing tissue. Analysing the DNA binding motives present in the proximal regulatory regions of genes down-regulated after wnt1 (RNAi) we found a few genes containing a TCF binding site, which include posterior Homeobox genes and chromatin remodelling proteins, suggesting that those are direct targets of the cWNT pathway and the responsible to trigger the expression of the posterior effectors. Furthermore, we have identified FoxG as an up-stream regulator of wnt1 transcription, probably though binding to an enhancer found in its first intron. Silencing of foxG inhibits the early phase of wnt1 expression and phenocopies the wnt1 (RNAi) phenotype, indicating its early role in specifying posterior versus anterior identity. Moreover, we have created a new open platform to interpret all transcriptomic and genomic results obtained (https://compgen.bio.ub.edu/PlanNET/planexp).

developmental biology↗

Asthma-associated variants induce IL33 differential expression through a novel regulatory region

Genome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as a strong regulatory element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. Supporting this notion, we show that genotype at an asthma-associated SNP, rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a novel mechanism through which a regulatory SNP contributes to genetic risk of asthma.

genomics↗

CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression

CTCF is an 11-zinc-finger DNA-binding protein that acts as a transcriptional repressor and insulator as well as an architectural protein required for 3D genome folding1-5. CTCF mediates long-range chromatin looping and is enriched at the boundaries of topologically associating domains, which are sub-megabase chromatin structures that are believed to facilitate enhancer-promoter interactions within regulatory landscapes 6-12. Although CTCF is essential for cycling cells and developing embryos13,14, its in vitro removal has only modest effects over gene expression5,15, challenging the concept that CTCF-mediated chromatin interactions and topologically associated domains are a fundamental requirement for gene regulation16-18. Here we link the loss of chromatin structure and gene regulation in an in vivo model and during animal development. We generated a ctcf knockout mutant in zebrafish that allows us to monitor the effect of CTCF loss of function during embryo patterning and organogenesis. CTCF absence leads to loss of chromatin structure in zebrafish embryos and affects the expression of thousands of genes, including many developmental genes. In addition, chromatin accessibility, both at CTCF binding sites and cis-regulatory elements, is severely compromised in ctcf mutants. Probing chromatin interactions from developmental genes at high resolution, we further demonstrate that promoters fail to fully establish long-range contacts with their associated regulatory landscapes, leading to altered gene expression patterns and disruption of developmental programs. Our results demonstrate that CTCF and topologically associating domains are essential to regulate gene expression during embryonic development, providing the structural basis for the establishment of developmental gene regulatory landscapes.

developmental biology↗

The Shh/Gli3 gene regulatory network precedes the origin of paired fins and reveals the deep homology between distal fins and digits.

One of the central problems of vertebrate evolution is understanding the relationship among the distal portions of fins and limbs. Lacking comparable morphological markers of these regions in fish and tetrapods, these relationships have remained uncertain for the past century and a half. Here we show that Gli3 functions in controlling the proliferative expansion of distal progenitors are shared among median and paired fins as well as tetrapod limbs. Mutant knockout gli3 fins in medaka (Oryzias latipes) form multiple radials and rays, in a pattern reminiscent of the polydactyly observed in Gli3 null mutant mice. In limbs, Gli3 controls both anterior-posterior patterning and cell proliferation, two processes that can be genetically uncoupled. In situ hybridization, quantification of proliferation markers, and analysis of regulatory regions reveal that in paired and median fins, gli3 plays a main role in controlling proliferation but not in patterning. Moreover, gli3 downregulation in shh mutant fins rescues fin loss in a manner similar to how Gli3-deficiency restores digits in the limbs of Shh mutant mouse embryos. We hypothesize that the Gli3/Shh pathway preceded the origin of paired appendages and was originally involved in modulating cell proliferation. Accordingly, the distal regions of median fins, paired fins, and limbs retain a deep regulatory and functional homology that predates the origin of paired appendages.

evolutionary biology↗

Boundary sequences flanking the mouse tyrosinase locus ensure faithful pattern of gene expression

Control of gene expression is dictated by cell-type specific regulatory sequences that physically organize the structure of chromatin, including promoters, enhancers and insulators. While promoters and enhancers convey cell-type specific activating signals, insulators prevent the cross-talk of regulatory elements within adjacent loci and safeguard the specificity of action of promoters and enhancers towards their targets in a tissue specific manner. Using the mouse tyrosinase (Tyr) locus as an experimental model, a gene whose mutations are associated with albinism, we described the chromatin structure in cells at two distinct transcriptional states. Guided by chromatin structure, through the use of Chromosome Conformation Capture (3C), we identified sequences at the 5 and 3 boundaries of this mammalian gene that function as enhancers and insulators. By CRISPR/Cas9-mediated chromosomal deletion, we dissected the functions of these two regulatory elements in vivo in the mouse, at the endogenous chromosomal context, and proved their role as genomic insulators, shielding the Tyr locus from the expression patterns of adjacent genes.

genomics↗

Cis-regulatory similarities in the zebrafish and human pancreas uncover potential disease-related enhancers

Introductory paragraphThe pancreas is a central organ for human diseases that have a dramatic societal burden, such as pancreatic cancer and diabetes1,2. Non-coding cis-regulatory elements (CREs) of DNA control gene expression3,4, being required for proper pancreas function. Most disease-associated alleles5,6 are non-coding, often overlapping with CREs5, suggesting that alterations in these regulatory sequences contribute to human pancreatic diseases by impairing gene expression. However, functional testing of CREs in vivo is not fully explored. Here we analysed histone modifications, transcription, chromatin accessibility and interactions, to identify zebrafish pancreas CREs and their human functional equivalents, uncovering disease-associated sequences across species. We found a human pancreatic enhancer whose deletion impairs the tumour suppressor gene ARID1A expression, conferring a potential tumour suppressor role to this non-coding sequence. Additionally, we identified a zebrafish ptf1a distal enhancer which deletion generates pancreatic agenesis, demonstrating the causality of this condition in humans7 and the interspecies functional equivalency of enhancers.

genomics↗

Gain of gene regulatory network interconnectivity at the origin of vertebrates

Signaling pathways control a large number of gene regulatory networks (GRNs) during animal development, acting as major tools for body plan formation1. Remarkably, in contrast to the large number of transcription factors present in animal genomes, only a few of these pathways operate during development2. Moreover, most of them are largely conserved along metazoan evolution3. How evolution has generated a vast diversity of animal morphologies with such a limited number of tools is still largely unknown. Here we show that gain of interconnectivity between signaling pathways, and the GRNs they control, may have played a critical contribution to the origin of vertebrates. We perturbed the retinoic acid, Wnt, FGF and Nodal signaling pathways during gastrulation in amphioxus and zebrafish and comparatively examined its effects in gene expression and cis-regulatory elements (CREs). We found that multiple developmental genes gain response to these pathways through novel CREs in the vertebrate lineage. Moreover, in contrast to amphioxus, many of these CREs are highly interconnected and respond to multiple pathways in zebrafish. Furthermore, we found that vertebrate-specific cell types are more enriched in highly interconnected genes than those tissues with more ancestral origin. Thus, the increase of CREs in vertebrates integrating inputs from different signaling pathways probably contributed to gene expression complexity and the formation of new cell types and morphological novelties in this lineage.

evolutionary biology↗

Regulatory architecture of the RCA gene cluster captures an intragenic TAD boundary and enhancer elements in B cells

The Regulators of Complement Activation (RCA) gene cluster comprises several tandemly arranged genes with shared functions within the immune system. RCA members, such as complement receptor 2 (CR2), are well-established susceptibility genes in complex autoimmune diseases. Altered expression of RCA genes has been demonstrated at both the functional and genetic level, but the mechanisms underlying their regulation are not fully characterised. We aimed to investigate the structural organisation of the RCA gene cluster to identify key regulatory elements that influence the expression of CR2 and other genes in this immunomodulatory region. Using 4C, we captured extensive CTCF-mediated chromatin looping across the RCA gene cluster in B cells and showed these were organised into two topologically associated domains (TADs). Interestingly, an inter-TAD boundary was located within the CR1 gene at a well-characterised segmental duplication. Additionally, we mapped numerous gene-gene and gene-enhancer interactions across the region, revealing extensive co-regulation. Importantly, we identified an intergenic enhancer and functionally demonstrated this element upregulates two RCA members (CR2 and CD55) in B cells. We have uncovered novel, long-range mechanisms whereby autoimmune disease susceptibility may be influenced by genetic variants, thus highlighting the important contribution of chromatin topology to gene regulation and complex genetic disease. SIGNIFICANCEThe complement system is a complex network of protein effectors and regulators that play a key role in immunity. Several regulators of complement response are clustered within Regulators of Complement Activation (RCA) gene family. Its members are all functionally, structurally, and genetically related. However, the functional relevance of this close gene organisation is unknown. We show that the clustering of the RCA members is due to shared long-range regulatory elements and physical chromatin looping. We also reveal that the RCA genes are divided into two adjacent chromatin domains and a domain boundary falls within the body of an expressed gene (CR1). Overall, our findings in the RCA cluster offer insights into their evolution, biology and roles in disease.

genetics↗